Bone marrow stromal cells comprising a nucleic acid encoding a notch intracellular domain

ABSTRACT

There is provided a method of inducing differentiation of bone marrow stromal cells to neural cells or skeletal muscle cells by introduction of a Notch gene. Specifically, the invention provides a method of inducing differentiation of bone marrow stromal cells to neural cells or skeletal muscle cells in vitro, which method comprises introducing a Notch gene and/or a Notch signaling related gene into the cells, wherein the finally obtained differentiated cells are the result of cell division of the bone marrow stromal cells into which the Notch gene and/or Notch signaling related gene have been introduced. The invention also provides a method of inducing further differentiation of the differentiation-induced neural cells to dopaminergic neurons or acetylcholinergic neurons. The invention yet further provides a treatment method for neurodegenerative and skeletal muscle degenerative diseases which employs neural precursor cells, neural cells or skeletal muscle cells produced by the method of the invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.10/503,816, filed May 24, 2005, now U.S. Pat No. 7,682,825 which is a371 Application of PCT/JP03/01260, filed Feb. 6, 2003, which claimspriority to Japanese Patent Application No. 2002-30003, filed Feb. 6,2002. All of the above-referenced applications are incorporated hereinin their entireties.

TECHNICAL FIELD

The present invention relates to a method of inducing differentiation ofbone marrow stromal cells to neural precursor cells or neural cells, andespecially dopaminergic neurons, or to skeletal muscle cells byintroduction of a Notch gene, and further relates to neural precursorcells, neural cells or skeletal muscle cells obtained by the method andto the therapeutic use of the cells and a treatment method.

BACKGROUND ART

Reconstruction of neural function in advanced neurodegenerativeconditions such as Alzheimer's disease, Parkinson's disease, ALS(amyotrophic lateral sclerosis) and the like requires replacement of theneural cells lost by cell death. Although neural cell transplantationhas been attempted in animal experiments using embryonic or adult neuralstem cells, ES cells and embryonic neural cells, such uses face majorhurdles against their application in humans. Ethical issues surround theuse of embryonic stem cells or neural cells, and the question ofguaranteeing a stable supply is also a concern. The demonstrated abilityof ES cells to differentiate is currently attracting much attention, butin addition to the numerous ethical issues, the cost and labor requiredto induce differentiation to specific cell types and the risk of formingteratoid tumors after transplantation are factors impeding stableapplication of this technology. In order to use adult neural stem cells,they must be extracted by craniotomy since they are found in a verylimited core section of the central nervous system, and thus patientsundergoing regenerative treatment are also exposed to a tremendous riskand burden.

Although approximately 10 years have passed since isolation of centralnervous system stem cells in vitro, it has not yet been possible by thecurrently accepted protocols to differentiate neural stem cells andobtain large amounts of functional dopaminergic or cholinergic neurons(Lorenz Studer, Nature Biotechnology Dec. Issue, p. 117 (2001).

A research group led by Professors Samuel Weiss of Calgary University(Canada) and Tetsuro Shingo has achieved success in efficiently inducingdifferentiation of dopamine-producing neural cells by administering amixture of several tyrosine hydroxylase inducing factors (TH cocktail)into mice brains, but no previous example exists of inducingdifferentiation of dopaminergic neurons and cholinergic neurons frombone marrow stromal cells as according to the present invention.

Motor neurons are acetylcholinergic, and their application to suchintractable diseases as ALS (amyotrophic lateral sclerosis) has beenconsidered. In ALS, death of spinal marrow motor neurons for reasons asyet unknown leads to loss of muscle controlling nerves, therebypreventing movement of muscles throughout the body including therespiratory muscles, and leading to death of the patient within 2-3years after onset. Currently, no effective treatment exists for thiscondition, but rat ALS models are being established.

Most degenerative muscular diseases such as muscular dystrophy areprogressive, and therefore transplantation of skeletal muscle cells mayconstitute an effective treatment. In healthy individuals, satellitecells present in muscle tissue supplement for skeletal muscle that haslost its regenerative capacity, but in progressive muscular diseases thenumber of such cells is reduced and regenerative capacity is accordinglylower. Thus, while transplantation of skeletal muscle or its precursorcells can be used as treatment, no effective curative means yet exists.

In the course of development of the central nervous system, neurons andglial cells are induced to differentiate from relatively homogeneousneural precursor cells or neural stem cells. A mechanism is in placewhereby some of the cells in the precursor cell population differentiateto certain cell subtypes in response to differentiation signals, whilethe other cells remain undifferentiated. Specifically, previouslydifferentiated cells send out certain signals to their surrounding cellsto prevent further differentiation to cells of their own type. Thismechanism is known as lateral inhibition. In Drosophila, cells alreadydifferentiated to neurons express the “Delta” ligand while theirsurrounding cells express the Delta receptor “Notch”, and binding of theligand with receptor ensures that the surrounding cells do notdifferentiate to neural cells (Notch signaling). The Delta-Notch systemappears to function in spinal cord cells as well (see, for example,Chitnis, A., Henrique, D., Lewis, J., Ish-Horowicz, D., Kintner, C.:Nature, 375, 761-766 (1995)).

It is thought that cellular interaction via the membrane protein Notchplays a major role in the development process whereby a homogeneous cellgroup produces many diverse types, and specifically, that upon ligandstimulation by adjacent cells, Notch induces expression of HES1 or HES5which inhibit bHLH (basic helix-loop-helix) neurodifferentiation factorssuch as Mash1, Math1 and neurogenin, to suppress differentiation to thesame cell type as the adjacent cell (see, for example, Kageyama et al.,Saibo Kogaku [Cell Engineering] Vol. 18, No. 9, 1301-1306 (1999)).

The Notch intracellular pathway is currently understood as follows. WhenNotch is first activated by ligands on the surface of adjacent cells(Delta, Serrate, Jagged), its intracellular domain is cleaved off(Artavanis-Tsakonas S. et al.: Science (1999) 284:770-776 and Kageyamaet al., Saibo Kogaku [Cell Engineering] Vol. 18, No. 9, 1301-1306(1999)). After cleavage of the intracellular domain of Notch, itmigrates from the cell membrane to the nucleus with the help of anuclear localization signal (NLS) and in the nucleus forms a complexwith the DNA-binding protein RBP-Jκ (Honjo T.: Genes Cells (1996) 1:1-9and Kageyama et al., Saibo Kogaku [Cell Engineering] Vol. 18, No. 9,1301-1306 (1999)). RBP-Jκ itself is a DNA-binding repressor oftranscription, and in the absence of activated Notch it binds to thepromoter of the HES1 gene, which is a differentiation inhibiting factor,thereby blocking its expression; however, once the complex forms betweenRBP-Jκ and the intracellular domain of Notch, the complex acts insteadto activate transcription of the HES1 gene (see Jarriault S. et al.:Nature (1995) 377:355-358, Kageyama R. et al.: Curr. Opin. Genet. Dev.(1997) 7:659-665 and Kageyama et al., Saibo Kogaku [Cell Engineering]Vol. 18, No. 9, 1301-1306 (1999)). This results in expression of HES1and HES1-induced suppression of differentiation. In other words, Notchis believed to suppress differentiation via HES1 (see Kageyama et al.,Saibo Kogaku [Cell Engineering] Vol. 18, No. 9, 1301-1306 (1999)).

In mammals as well, it has become clear that Notch-mediated regulationof gene expression is important in maintaining neural precursor cells orneural stem cells and in the highly diverse process of neuraldifferentiation, and that the Notch pathway is also essential fordifferentiation of cells other than those of the nervous system (seeTomita K. et al.: Genes Dev. (1999) 13:1203-1210 and Kageyama et al.,Saibo Kogaku [Cell Engineering] Vol. 18, No. 9, 1301-1306 (1999)). Inaddition, the existence of a HES-independent Notch pathway, negativeregulation of Notch signaling on the transcription level and negativeinteraction on the protein level have also been anticipated (see Goh,M., Saibo Kogaku [Cell Engineering] Vol. 18, No. 9, 1291-1300 (1999)).Still, all of the aforementioned publications either teach or suggestthat Notch signaling acts in a direction which suppressesdifferentiation.

Central nervous disorders in which reconstruction is not an optionactually include a variety of different conditions with a high incidencerate in the population, from injury-induced spinal damage orcerebrovascular impairment or glaucoma which leads to blindness, toneurodegenerative conditions such as Parkinson's disease. Research onneuroregenerative methods to treat such diseases is therefore an urgentsocial need, and the results of this research by the present inventorsis believed to be a breakthrough for application to humans. Bone marrowstromal cells are easily extracted by bone marrow aspiration on anoutpatient basis, and due to their highly proliferative nature they canbe cultured in large amounts within a relatively short period. Moreovera tremendous advantage may be expected since autologous transplantationcan be carried out if nerves are formed from one's own bone marrow stemcells. The lack of immunological rejection would dispense with the needfor administering immunosuppressants, thus making safer treatmentpossible. Furthermore, since bone marrow stem cells can be obtained froma bone marrow bank, this method is realistically possible from a supplystandpoint. If such cells can be used to derive neural cells, for whichno effective means has heretofore existed, then a major effect may beexpected in the field of regenerative medicine.

ALS (amyotrophic lateral sclerosis) is a condition in which cell deathof spinal marrow motor neurons for reasons as yet unknown leads to lossof muscle controlling nerves, thereby preventing movement of musclesthroughout the body including the respiratory muscles and leading todeath of the patient within 2-3 years after onset, but at the currenttime no effective treatment exists. Formation of acetylcholinergicneurons from one's own bone marrow stem cells would allow autologoustransplantation, and this would offer a major benefit that might evenserve as a cure for ALS.

Effective treatment methods also currently do not exist for musculardiseases such as muscular dystrophy, a degenerative disease of theskeletal muscle. A major benefit would also be afforded for suchconditions, since formation of skeletal muscle cells from one's own bonemarrow stem cells would allow autologous transplantation. Using suchcells to derive skeletal muscle cells, for which no effective means hasheretofore existed, would also be expected to provide a major effect inthe field of regenerative medicine.

The possible applications of this technology are not only in the fieldof clinical treatment but also in the area of engineering of artificialorgans and the like, which is expected to be an important field ofdevelopment in the future. If neural cells or muscle cells could beeasily produced on a cell culturing level, then applications may beimagined for creation of hybrid artificial organs and the like.

BRIEF DESCRIPTION OF THE INVENTION

The present invention provides a method of inducing differentiation ofbone marrow stromal cells to neural cells or skeletal muscle cells invitro, which method comprises introducing a Notch gene and/or a Notchsignaling related gene into the cells, wherein the finally obtaineddifferentiated cells are the result of cell division of the bone marrowstromal cells into which the Notch gene and/or Notch signaling relatedgene have been introduced. The invention further provides a noveltreatment method for neurodegenerative and skeletal muscle degenerativediseases which employs neural precursor cells, neural cells or skeletalmuscle cells obtained by the aforementioned method.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a micrograph (phase contrast microscope) in lieu of a drawing,showing neural cells induced to differentiate according to theinvention.

FIG. 2 is a composite of immunofluorescent photographs in lieu of adrawing, showing positive reactions of neural cells induced todifferentiate according to the invention, against MAP-2 antibodies,neurofilament antibodies and nestin antibodies.

FIG. 3 is a composite of immunofluorescent photographs in lieu of adrawing, showing reactions of neural cells induced to differentiateaccording to the invention, against antibodies for the neurotransmittersynthetase tyrosine hydroxylase (TH) and the neurotransmitters orneurotransmitter-related peptides vesicular acetylcholine transporter(VAChT), neuropeptide Y (NPY), substance P (SP), glutamine (Glu),calcitonin gene related peptide (CGRP) and vasoactive intestinal peptide(VIP).

FIG. 4 is a pair of immunofluorescent photographs in lieu of a drawing,showing changes in the tyrosine hydroxylase positivity (rate ofdopaminergic neuron differentiation) of neural cells induced todifferentiate according to the invention, before and after treatmentwith GDNF.

FIG. 5 is a graph showing changes in the tyrosine hydroxylase positivity(rate of dopaminergic neuron differentiation) of neural cells induced todifferentiate according to the invention, before and after treatmentwith GDNF.

FIG. 6 is a pair of immunofluorescent photograph in lieu of a drawing,showing changes in the vesicular acetylcholine transporter positivity(rate of acetylcholinergic neuron differentiation) of neural cellsinduced to differentiate according to the invention, before and aftertreatment with neurotrophins (NTs; 2.5 S NGF).

FIG. 7 is a graph showing changes in the vesicular acetylcholinetransporter positivity (rate of acetylcholinergic neurondifferentiation) of neural cells induced to differentiate according tothe invention, before and after treatment with neurotrophins (NTs; 2.5 SNGF).

FIG. 8 is a micrograph (phase contrast microscope) in lieu of a drawing,showing skeletal muscle cells induced to differentiate according to theinvention.

FIG. 9 is another micrograph (phase contrast microscope) in lieu of adrawing, showing skeletal muscle cells induced to differentiateaccording to the invention. This photograph shows the increase in theskeletal muscle of FIG. 8 with time.

FIG. 10 is a confocal laser micrograph in lieu of a drawing, showing thepolynucleated nature of skeletal muscle cells induced to differentiateaccording to the invention. The nuclei and the actin filaments can beseen.

FIG. 11 is a pair of graphs showing the therapeutic effect oftransplanting dopaminergic neurons obtained by the differentiationinducing method of the invention into striata of rat Parkinson's diseasemodels.

FIG. 12 is a composite of immunofluorescent photographs in lieu of adrawing, showing that the cells transplanted into the striata were notglial cells but neural cells and dopaminergic neurons.

FIG. 13 is a composite of magnified immunofluorescent photographs inlieu of a drawing, showing that the cells transplanted into the striatawere neural cells and dopaminergic neurons.

FIGS. 14 a to 14 f show the features of isolated bone marrow stromalcells (MSC). FIG. 14 a shows FACS analysis results for rat (MSC). Thecells expressed CD29 (β1-integrin), CD90 (Thy-1) and CD54 (ICAM), butnot CD34 (hemopoietic stem cell marker) or CD11b/c (macrophage-relatedmarker). FIGS. 14 b and 14 c are phase contrast micrographs ofnon-treated rat MSCs (b) and non-treated human MSCs (c). FIGS. 14 d to14 f are immunohistochemical photographs of CD29 (d), CD90 (e) and CD34(f) in human MSCs. The MSCs were positive for CD29 and CD90, butnegative for CD34. The bar represents 50 μm.

FIGS. 15 a to 15 h show phenotypes after NICD (Notch intracellulardomain) transfection. FIG. 15 a shows the results of RT-PCR for theNotch extracellular domain (ECD) and intracellular domain (ICD) in ratMSC, before NICD transfection (lane 1) and after NICD transfection (lane2). Since ECD was detected in the non-treated MSC, a small amount ofendogenous Notch was naturally expressed. After NICD transfection,however, ECD was down-regulated and NICD slightly up-regulated. FIGS. 15b to 15 g are immunohistochemical photographs for GLAST (b, c), 3-PGDH(d, e) and nestin (f, g) in non-treated rat MSCs (b, d, f) andNICD-transfected rat MSCs (c, e, g). The bar represents 50 μm in b, c, dand g and 80 μm in e and f. FIG. 15 h is a graph showing 3-PGDH promoteractivity for non-treated rat MSCs (MSC) and NICD-transfected rat MSCs(NICD). Both the full-length form of 3-PGDH and the truncated form(M1965) showed 9- to 10-fold increases in promoter activity after NICDtransfection (p<0.01).

FIG. 16 shows the rates of conversion to MAP-2ab⁺ cells upon treatmentwith various trophic factors. No MAP-2ab⁺ cells were detected when thetrophic factors were introduced into rat MSCs either non-treated ortransfected with a control vector. Introduction of three of the trophicfactors (FSK+6FGF+CNTF) showed the highest rate of neural cellproduction (96.5%), while elimination of any of these three factorsresulted in a lower conversion rate.

FIGS. 17 a to 17 q show the analysis results for induced neural cells.FIGS. 17 a to 17 c are phase contrast micrographs of neural cellsinduced from rat MSCs (a, b) and human MSCs (c). The bar represents 200μm in FIGS. 17 a and 50 μm in FIGS. 17 b and 17 c. FIGS. 17 d to 17 gand 17 i to 17 k are immunohistochemical photographs of neuron markersand glia markers in rat MSCs (f, g, i, j, k) and human MSCs (d, e) (5days) after introduction of trophic factors. The markers MAP-2ab (d) andneurofilament-M (e) were detected in human MSC, while β3-tubulin (f) andTuJ-1 (g) were expressed in rat MSCs. None of the rat or human cellsreacted with the glia markers GFAP (i), GalC (j) and O4 (k). The barrepresents 100 μm in d, e and f, 60 μm in g and 100 μm in i to k. FIG.17 h shows Brd-U labeling of neural cells. MAP-2ab positive cells (AlexaFluor 488-labeled, green code) did not incorporate Brd-U (Alexa Fluor546-labeled, red code). FIG. 17 l shows Western blot analysis resultsfor the MAP-2ab rat sample (1) and GFAP rat sample (2). Lane 1 isWestern blotting and lane 2 is a ponceau S stain. The non-treated MSCs(M) expressed neither MAP-2ab nor GFAP. On the 5th day (N) afterintroducing the trophic factors, the MSCs were MAP-2ab positive but werestill negative for GFAP. Brain (B) was used as a positive control forboth MAP-2ab and GFAP. FIGS. 17 m to 17 q show the results of a patchclamp test with neural cells induced from rat MSCs (m) and neural cellsinduced from human MSCs (n, p). Induction resulted in a dramaticincrease in rectified K⁺ current up to approximately 1600 pA and 4000 pAin the rat MSCs (m) and human MSCs (n), respectively, compared to thenon-treated MSCs (o, p). FIG. 17 q shows a phase contrast micrograph ofhuman MSCs recorded in FIG. 17 n.

FIG. 18 is a pair of graphs showing relative promoter activities ofNeuro D and GFAP for non-treated rat MSCs (MSC), NICD-transfected ratMSCs (NICD) and neural-induced rat MSCs (induced).

FIGS. 19 a to 19 m show the results of transplantation into ratParkinson's disease models. FIG. 19 a is a graph showing the percentagesof the following neurotransmitters in rat MSCs after trophic factorinduction: γ-aminobutyric acid (GABA); 0.3±0.1, vasoactive intestinalpeptide (VIP); 0.5±0.1, serotonin (Ser); 2.0±0.4, glutamate (Glu);2.3±0.7, substance P (SP); 2.4±0.9, TH; 3.9±0.6, vesicular acetylcholinetransporter (VACht); 5.2±2.4, calcitonin gene related peptide (CGRP);5.3±0.8, neuropeptide Y (NPY)6.1±1.6. With subsequent administration ofGDNF, the percentage of TH-positive cells increased drastically to41.0±14.1 (G-TH). FIGS. 19 b and 19 c show TH expression in human MSCsafter trophic factor induction and then after GDNF treatment. The humanMSCs exhibited the same response as rat MSCs, with TH-positive cellsclearly increasing after GDNF treatment. The bar represents 100 μm in band 30 μm in c. FIG. 19 d shows the results or RT-PCR of Nurr-1 in ratMSCs. Increased Nun-1 up-regulation was observed after administration ofGDNF (Ng) compared to the cells after trophic factor introduction alone(N). FIG. 19 e shows a Western blot for TH in rat MSCs. TH expressionwas weak in MSCs after trophic factor induction (N), but increased afterGDNF induction (Ng). Adrenal medulla (A) served as a positive control.Lane 1 is Western blotting and lane 2 is a ponceau S stain. FIG. 19 f isa graph showing behavioral effects after grafting rat MSCs into thestriatum. The graph shows apomorphine-induced rotation in an MSC group(▴-▴), N-MSC group (●-●) and G-MSC group (▪-▪) (*:0.01<p<0.05;**:p<0.01). FIGS. 19 g to 19 k are immunostaining photographs forneurofilament-M (g), TH (h), DAT (i), GFAP (j) and O4 (i) in striatum atthe 10th week after transplantation into the G-MSC group. Signals forthese markers were all labeled with Alexa 546. The grafted rat MSCs werefirst labeled with GFP. Doubling of GFP-neurofilament, GFP-TH andGFP-DAT was observed in g, h and i, but not with GFAP staining or O4staining. The bar represents 50 μm. FIG. 19 l is a set of sectionalillustrations showing integration of GFP-labeled rat MSCs (G-MSC group)into the striatum. Confocal images after immunohistochemistry for TH areindicated from regions marked by dots in the diagram. The bar represents50 μm. FIG. 19 m is a graph showing apomorphine induced rotation in ratsafter transplantation of GDNF-treated human neural MSCs. The resultsfrom 5 rats (mean rotation: 0.44±0.2) are shown up to four weeks aftergrafting (with one rat represented by each line).

DETAILED DESCRIPTION OF THE INVENTION

The present inventors investigated stimulation of bone marrow stromalcells by introduction of genes which play a central role in the initialstages of morphogenesis of bone marrow stromal cells, and examined theeffects of such stimulation on induction of bone marrow stromal celldifferentiation. Specifically, it was expected to be potentiallypossible to “reset” bone marrow stromal cells by introduction of Notchgenes and Notch signaling genes, which play important roles indevelopmental differentiation of the nervous system and performfunctions in determining cell fates when precursor cells branch toneural cells or glial cells.

It is important to note that despite implication of Notch genes andNotch signaling related genes in the mechanism of suppressing inductionof cell differentiation, it was a completely unexpected finding thatcombining introduction of Notch genes and Notch signaling related geneswith other stimulation to induce differentiation, can also inducedifferentiation of the very cells into which the Notch genes and Notchsignaling related genes have been introduced (not the cells contactingwith the cells into which the Notch genes and Notch signaling relatedgenes have been introduced). It cannot be affirmed that introduction ofthe Notch genes and Notch signaling related genes in the differentiationinducing method of the present invention resulted in resetting ofdevelopmental differentiation of bone marrow stromal cells. However, bycombination of this gene introduction with other differentiationinducing steps according to the invention, it was possible as a resultto provide a method of efficiently inducing differentiation of bonemarrow stromal cells to neural cells or skeletal muscle cells.

As a result of repeated experimentation in combining steps comprisingintroduction of Notch genes and Notch signaling related genes, thepresent inventors have been the first to succeed in efficiently inducingdifferentiation of bone marrow stromal cells to neural cells or skeletalmuscle cells in vitro. Moreover, it was confirmed that upon grafting ofthe neural cells obtained by the differentiation inducing method intorat Parkinson's disease models or rat optic nerve damage-associatedretinal or optic nerve degeneration models, the grafted nerves actuallytook and functioned, and the present invention was thus completed.

Surprisingly, by introducing Notch genes and Notch signaling relatedgenes into bone marrow stromal cells, by administration of variousfactors and cytokines believed to be involved in promoting neuraldifferentiation, and by increasing intracellular cAMP which isconsidered to be a general trigger for initiation of differentiation, itwas possible to successfully induce differentiation of bone marrowstromal cells to neural cells under in vitro culturing conditions. Weconfirmed not only expression of MAP-2 and neurofilament which arespecific to neural cells, but also expression of the neurotransmittersynthetase tyrosine hydroxylase and production of neurotransmitters suchas acetylcholine, neuropeptide Y and substance P.

On the other hand, it has been suggested that demethylation andactivation of one or a very few genes by 5-azacytidine (5-AZC) leads toconversion to myoblasts (see Taylar S M, Jones P A: Cell 17:771-779,1979 and Nabeshima Y., Seitai no Kagaku 47(3):184-189, 1996). Wetherefore combined the aforementioned introduction of Notch genes andNotch signaling related genes into neural cells with the aforementioneddemethylation by treatment with 5-azacytidine (5-AZC). Specifically, byeliminating suppressed expression by methylation of the genes using theaforementioned demethylating agent to reset bone marrow stromal cells,subsequently introducing the Notch and Notch signaling related genes andco-culturing the gene-introduced cells together with bone marrow stromalcells without the genes, and finally treating the cells with anaugmenting agent for intracellular cAMP which is considered to be ageneral trigger for initiating differentiation, we succeeded in inducingdifferentiation of the Notch and Notch signaling related gene-introducedcells to skeletal cells by culturing in vitro. Characteristicpolynucleated myotube formation and striation were found in theresultant cells, and expression of the muscle-specific proteins myogeninand Myf5 was also confirmed on the mRNA level.

According to one mode of the invention, there is provided a method ofinducing differentiation of bone marrow stromal cells to neural cells orskeletal muscle cells in vitro, which method comprises introducing aNotch gene and/or a Notch signaling related gene into the cells, whereinthe resultant differentiated cells are the offspring of cell division ofthe bone marrow stromal cells into which the Notch gene and/or Notchsignaling related gene have been introduced.

According to another mode of the invention, there is provided a methodof inducing bone marrow stromal cells to differentiate into neuralprecursor cells in vitro comprising the steps of:

(1) isolating bone marrow stromal cells from bone marrow, and culturingthe cells in a standard essential culture medium supplemented with aserum; and

(2) introducing a Notch gene and/or a Notch signaling related gene intothe cells, and further culturing the calls to produce neural precursorcells.

The isolated bone marrow stromal cells may be human cells.

According to yet another mode of the invention, there are providedneural precursor cells produced by the aforementioned method.

According to yet another mode of the invention, there are providedneural precursor cells which express the neural precursor cell markersGLAST, 3PGDH and nestin.

According to yet another mode of the invention, there is provided amethod of inducing bone marrow stromal cells to differentiate intoneural cells in vitro comprising the steps of:

(1) isolating bone marrow stromal cells from bone marrow, and culturingthe cells in a standard essential culture medium supplemented with aserum;

(2) introducing a Notch gene and/or a Notch signaling related gene intothe cells, and further culturing the calls; and

(3) adding a cyclic AMP-augmenting agent or a cyclic AMP analogue,and/or a cell differentiation stimulating factor to the culture medium,and further culturing the cells to produce the neural cells,

wherein the resultant differentiated cells are offspring of celldivision of the bone marrow stromal cells into which the Notch geneand/or Notch signaling related gene have been introduced.

The standard essential culture medium may be an Eagle's alpha modifiedminimum essential medium, and the serum may be fetal bovine serum.

The introduction of the Notch gene and/or Notch signaling related genemay be accomplished by lipofection with a mammalian expression vector.

The method may also comprise, between steps (2) and (3), a step ofselecting cells into which the genes have been introduced, for apredetermined period of time.

The cyclic AMP-augmenting agent or cyclic AMP analogue may be forskolin,and its concentration may be 0.001 nM to 100 μM.

The cell differentiation stimulating factor may be selected from thegroup consisting of basic fibroblast growth factor (bFGF), ciliaryneurotrophic factor (CNTF) and mixtures thereof.

The concentration of the cell differentiation stimulating factor may bebetween 0.001 ng/ml and 100 μg/ml.

The isolated bone marrow stromal cells are preferably human cells.

According to yet another mode of the invention, there are providedneural cells produced by the aforementioned method.

According to yet another mode of the invention, there are providedneural cells which express the neural cell markers β-tubulin isotype 3and TuJ-1.

According to yet another mode of the invention, there is provided amethod of inducing bone marrow stromal cells to differentiate intodopaminergic neurons in vitro comprising the steps of:

(1) isolating bone marrow stromal cells from bone marrow, and culturingthe cells in a standard essential culture medium supplemented with aserum;

(2) introducing a Notch gene and/or a Notch signaling related gene intothe cells, and further culturing the cells;

(3) adding a cyclic AMP-augmenting agent or a cyclic AMP analogue,and/or a cell differentiation stimulating factor to the culture medium,and further culturing the cells to produce the neural cells;

(4) culturing the neural cells obtained in Step (3) in a standardessential culture medium supplemented with a serum; and

(5) adding glial derived neurotrophic factor (GDNF), and a cyclicAMP-augmenting agent or a cyclic AMP analogue, and/or a celldifferentiation stimulating factor other than glial derived neurotrophicfactor to the culture medium, and further culturing the cells to obtaindopaminergic neurons,

wherein the resultant dopaminergic neurons are offspring of bone marrowstromal cells into which the Notch gene and/or Notch signaling relatedgene have been introduced.

The standard essential culture medium in Step (4) may be an Eagle'salpha modified minimum essential medium.

The serum in Step (4) may be fetal bovine serum.

The cyclic AMP-augmenting agent or cyclic AMP analogue in Step (5) maybe forskolin. The concentration of the cyclic AMP-augmenting agent orcyclic AMP analogue in Step (5) may be between 0.001 nM and 100 μM.

The cell differentiation stimulating factor other than glial derivedneurotrophic factor in Step (5) may be selected from the groupconsisting of basic fibroblast growth factor (bFG), platelet-derivedgrowth factor-AA (PDGF-AA) and mixtures thereof.

The concentration of glial derived neurotrophic factor in (Step 5) maybe between 0.001 ng/ml and 100 μg/ml, and is preferably between 1 ng/mland 100 ng/ml.

The concentration of the cell differentiation stimulating factor otherthan glial derived neurotrophic factor in Step (5) may be between 0.001ng/ml and 100 μg/ml.

The isolated bone marrow stromal cells are preferably human cells.

According to yet another mode of the invention, there are provideddopaminergic neurons produced by the aforementioned method.

According to yet another mode of the invention, there is provided amethod of inducing bone marrow stromal cells to differentiate intoacetylcholinergic neurons in vitro comprising the steps of:

(1) isolating bone marrow stromal cells from bone marrow, and culturingthe cells in a standard essential culture medium supplemented with aserum;

(2) introducing a Notch gene and/or a Notch signaling related gene intothe cells, and further culturing the cells;

(3) adding a cyclic AMP-augmenting agent or a cyclic AMP analogue,and/or a cell differentiation stimulating factor to the culture medium,and further culturing the cells to produce the neural cells;

(4) culturing the neural cells obtained in Step (3) in a standardessential culture medium supplemented with a serum; and

(5) adding nerve growth factor (NGF), and a cyclic AMP-augmenting agentor a cyclic AMP analogue, and/or a cell differentiation stimulatingfactor other than nerve growth factor to the culture medium, and furtherculturing the cells to obtain acetylcholinergic neurons,

wherein the resultant acetylcholinergic neurons are offspring of bonemarrow stromal cells into which the Notch gene and/or Notch signalingrelated gene have been introduced.

The standard essential culture medium in Step (4) may be an Eagle'salpha modified minimum essential medium. The serum in Step (4) may befetal bovine serum.

The cyclic AMP-augmenting agent or cyclic AMP analogue in Step (5) maybe forskolin. The concentration of the cyclic AMP-augmenting agent orcyclic AMP analogue in Step (5) may be between 0.001 nM and 100 μM.

The cell differentiation stimulating factor other than nerve growthfactor in Step (5) may be selected from the group consisting of basicfibroblast growth factor (bFG), platelet-derived growth factor-AA(PDGF-AA) and mixtures thereof.

The concentration of nerve growth factor in (Step 5) may be between0.001 ng/ml and 100 μg/ml, and is preferably between 1 ng/ml and 100ng/ml.

The concentration of the cell differentiation stimulating factor otherthan nerve growth factor in Step (5) may be between 0.001 ng/ml and 100μg/ml.

The isolated bone marrow stromal cells are preferably human cells.

According to yet another mode of the invention, there are providedacetylcholinergic neurons produced by the aforementioned method.

According to yet another mode of the invention, there is provided amethod of inducing bone marrow stromal cells to differentiate intoskeletal muscle cells in vitro, comprising the steps of:

(1) isolating bone marrow stromal cells from bone marrow, and culturingthe cells in a standard essential culture medium supplemented with aserum;

(2) adding a demethylating agent to the culture medium, and furtherculturing the cells;

(3) adding a cyclic AMP-augmenting agent or a cyclic AMP analogue,and/or a cell differentiation stimulating factor to the culture medium,and further culturing the cells;

(4) introducing a Notch gene and/or a Notch signaling related gene intothe cells, and further culturing the cells;

(5) co-culturing the cells into which the genes have been introduced,with non-treated bone marrow stromal cells into which the genes have notbeen introduced; and

(6) adding a cyclic AMP-augmenting agent or a cyclic AMP analogue to theculture medium, and further culturing the cells to obtain skeletalmuscle cells,

wherein the resultant differentiated cells are offspring of bone marrowstromal cells into which the Notch gene and/or Notch signaling relatedgene have been introduced.

The standard essential culture medium may be an Eagle's alpha modifiedminimum essential medium, and the serum may be fetal bovine serum.

The demethylating agent may be 5-azacytidine, and its concentration maybe between 30 nmol/l and 300 μmol/l.

The cyclic AMP-augmenting agent or cyclic AMP analogue in Step (3) maybe forskolin.

The concentration of the cyclic AMP-augmenting agent or cyclic AMPanalogue in Step (3) may be between 0.001 nM and 100 μM.

The cell differentiation stimulating factor may be selected from thegroup consisting of basic fibroblast growth factor (bFGF),platelet-derived growth factor-AA (PDGF-AA), heregulin, and mixturesthereof, and its concentration may be between 0.001 ng/ml and 100 μg/ml.The introduction of the Notch gene and/or Notch signaling related genemay be accomplished by lipofection with a mammalian expression vector.

The method may also comprise, between steps (4) and (5), a step ofselecting cells into which the genes have been introduced, for apredetermined period of time.

The cyclic AMP-augmenting agent or cyclic AMP analogue in Step (5) maybe forskolin.

The concentration of the cyclic AMP-augmenting agent or cyclic AMPanalogue in Step (5) may be between 0.001 nM and 100 μM.

The isolated bone marrow stromal cells are preferably human cells.

According to yet another mode of the invention, there are providedskeletal muscle cells produced by the aforementioned method.

According to yet another mode of the invention, there is provided amethod for treatment of a patient suffering from a disease, disorder orcondition of the central nervous system, which method comprisesadministering a therapeutically effective amount of the aforementionedneural precursor cells into the region of the central nervous system ofthe patient in which the disease, disorder or condition is found,wherein the presence of the neural precursor cells exerts a therapeuticeffect on the disease, disorder or condition.

According to yet another mode of the invention, there is provided theuse of a therapeutically effective amount of the aforementioned neuralprecursor cells in the manufacture of a pharmaceutical composition fortreatment of a patient suffering from a disease, disorder or conditionof the central nervous system.

According to yet another mode of the invention, there is provided amethod for treatment of a patient suffering from a disease, disorder orcondition of the central nervous system, which method comprisesadministering a therapeutically effective amount of the aforementionedneural cells into the region of the central nervous system of thepatient in which the disease, disorder or condition is found, whereinthe presence of the neural cells exerts a therapeutic effect on thedisease, disorder or condition.

According to yet another mode of the invention, there is provided theuse of a therapeutically effective amount of the aforementioned neuralcells in the manufacture of a pharmaceutical composition for treatmentof a patient suffering from a disease, disorder or condition of thecentral nervous system.

According to yet another mode of the invention, there is provided amethod for treatment of a patient suffering from a disease, disorder orcondition of the central nervous system, which method comprisesadministering a therapeutically effective amount of the aforementionedneural cells which express the neural cell markers β-tubulin isotype 3and TuJ-1 into the region of the central nervous system of the patientin which the disease, disorder or condition is found, wherein thepresence of the neural cells exerts a therapeutic effect on the disease,disorder or condition.

According to yet another mode of the invention, there is provided theuse of a therapeutically effective amount of the aforementioned neuralcells which express the neural cell markers β-tubulin isotype 3 andTuJ-1 in the manufacture of a pharmaceutical composition for treatmentof a patient suffering from a disease, disorder or condition of thecentral nervous system.

According to yet another mode of the invention, there is provided amethod for treatment of a patient suffering from a disease, disorder orcondition of the central nervous system, which method comprisesadministering a therapeutically effective amount of the aforementioneddopaminergic neurons into the region of the central nervous system ofthe patient in which the disease, disorder or condition is found,wherein the presence of the neural cells exerts a therapeutic effect onthe disease, disorder or condition.

According to yet another mode of the invention, there is provided theuse of a therapeutically effective amount of the aforementioneddopaminergic neurons in the manufacture of a pharmaceutical compositionfor treatment of a patient suffering from a disease, disorder orcondition of the central nervous system.

According to yet another mode of the invention, the disease, disorder orcondition may be Parkinson's disease.

According to yet another mode of the invention, there is provided amethod for treatment of a patient suffering from a disease, disorder orcondition of the central nervous system, which method comprisesadministering a therapeutically effective amount of the aforementionedacetylcholinergic neurons into the region of the central nervous systemof the patient in which the disease, disorder or condition is found,wherein the presence of the neural cells exerts a therapeutic effect onthe disease, disorder or condition.

According to yet another mode of the invention, there is provided theuse of a therapeutically effective amount of the aforementionedacetylcholinergic neurons in the manufacture of a pharmaceuticalcomposition for treatment of a patient suffering from a disease,disorder or condition of the central nervous system.

The disease, disorder or condition may be selected from the groupconsisting of ALS (amyotrophic lateral sclerosis) and Alzheimer'sdisease.

According to yet another mode of the invention, there is provided amethod for treatment of a patient suffering from a disease, disorder orcondition associated with muscle degeneration, which method comprisesadministering a therapeutically effective amount of the aforementionedskeletal muscle cells into the region of muscular degeneration of thepatient, wherein the presence of the skeletal muscle cells exerts atherapeutic effect on the disease, disorder or condition.

According to yet another mode of the invention, there is provided theuse of a therapeutically effective amount of the aforementioned skeletalmuscle cells in the manufacture of a pharmaceutical composition fortreatment of a patient suffering from a disease, disorder or conditionassociated with muscle degeneration.

The disease, disorder or condition may be muscular dystrophy.

Throughout the present specification, the term “bone marrow stromalcells” refers to cells in the bone marrow which are not of thehemopoietic system and are potentially able to differentiate toosteocytes, chondrocytes, adipocytes and the like. Bone marrow stromalcells are identified by positivity for CD29 (β1-integrin), CD90 (Thy-1)and CD54 (ICAM-1) and negativity for CD34 (hemopoietic stem cell marker)and CD11b/c (macrophage marker).

The term “efficiently” as used throughout the present specification withrespect to inducing differentiation means that the selected bone marrowstromal cells are finally converted to neural cells or skeletal musclecells at a high rate by the differentiation inducing method of theinvention. The efficiency of the differentiation inducing method of theinvention is 50% or greater, preferably 75% or greater, more preferably80% or greater, even more preferably 85% or greater, yet more preferably90% or greater and most preferably 95% or greater.

The term “neural precursor cells” as used throughout the presentspecification refers to bone marrow stromal cells immediately afterintroduction of a Notch gene and/or Notch signaling related gene, andspecifically they are the cells prior to introduction of trophicfactors.

The term “neural cells” as used throughout the present specificationrefers to neurons, which are characterized morphologically by a cellbody and two types of processes (dendrites and axons), and biochemicallyby reaction with antibodies for β-tubulin isotope 3 and TuJ-1.

Neural cells are characterized by secreting neurotransmitters,neurotransmitter synthetases or neurotransmitter-related proteins, forexample, tyrosine hydroxylase (TH), vesicular acetylcholine transporter,neuropeptide Y and substance P (SP).

Tyrosine hydroxylase is a marker for dopaminergic neurons, whilevesicular acetylcholine transporter is a marker for acetylcholinergicneurons which are typically motor neurons.

The term “glial cells” as used throughout the present specificationrefers to astrocytes, oligodendrocytes, microglia and epithelial cellsfound between neurons and their processes in the central nerves.

Glial fibrillar acidic protein (GFAP) is a marker for astrocytes, and O4is a marker for oligodendrocytes.

The term “skeletal muscle cells” as used throughout the presentspecification refers to myofibers or muscle fibers, and they are theindividual myocytes of the skeletal muscle. Morphologically they arecharacterized as giant long, thin polynucleated cells with myotubeformation and striation, while biochemically they are characterized byexpressing transcription regulating factors such as myogenin and Myf5.

The method of inducing differentiation of bone marrow stromal cells intoneural cells or skeletal muscle cells according to the invention isnovel in the aspect of comprising a step of introducing a Notch geneand/or Notch signaling related gene into the aforementioned cells.Another novel aspect is that this step may be combined with otherdifferentiation inducing steps of the prior art in a prescribed order.The selection and optimum combination of such steps according to theinvention constitute a highly significant novel discovery by the presentinventors. Bone marrow stromal cells had already been known asmesenchymal stem cells or precursor cells capable of being induced todifferentiate to osteoblasts, vascular endothelial cells, skeletalmuscle cells, adipocytes and smooth muscle cells, but it was not knownwhether bone marrow stromal cells could actually be differentiated toneural cells or skeletal muscle cells, and this goal had not yet beensuccessfully achieved despite vigorous attempts. While not intending tobe constrained by any particular theory, the present inventorsconjecture that introduction of a Notch gene and/or Notch gene signalingrelated gene into the aforementioned cells results in resetting of thecells in terms of developmental differentiation, and aid in the functionof other differentiation inducing treatments.

The present invention will now be explained in greater detail by thefollowing examples, with the understanding that these examples do notlimit the scope of the invention in any way.

EXAMPLES Example 1 Neural Induction

Stromal cells were extracted from the bone marrow of adult rats (Wistarrats) and cultured. The medium used was Minimum Essential Medium AlphaEagle Modification (M4526, Sigma Co.) containing 20% fetal bovine serum(14-501F, Lot #61-1012, BioWhittaker Co.).

After subculturing to four generations, the gene for the Notchintracellular domain was introduced when the cells reached 80-90%confluence. A 3.1 kb EcoRI-XbaI fragment of the Notch intracellulardomain was inserted at the EcoRI-XbaI multicloning site of pCI-neomammal expression vector (#E1841) by Promega for recombination. ALipofectAMINE 2000 (11668-027, Gibco BRL) system was used for theintroduction.

On the day following introduction, G418 sulfate (83-5027, Gibco BRL) wasadded to a concentration of 200 ng/ml and introduced cells were selectedfor 10 days.

After restoration of the cell population to 90% confluence, 5 μM offorskolin (344273, Calbiochem), 10 ng/ml of basic fibroblast growthfactor (100-18B, Peprotech EC, Ltd.) and 50 ng/ml of ciliaryneurotrophic factor (557-NT, R&D Systems) were added.

As a result of analyzing the cells after about 10 days, thecharacteristic morphology of neural cells was observed as shown inFIG. 1. The induced cells exhibited positive reaction for antibodiesagainst MAP-2 (MAB364, Chemicon), neurofilament (814342, BoehringerManheim) and nestin (BMS4353, Bioproducts), as shown in FIG. 2. SinceMAP-2 and neurofilament are markers for neural cells and nestin is amarker for neural precursor cells, the induced cells were thereforejudged to possess the properties of neural cells.

A search conducted using antibodies against the neurotransmittersynthetase tyrosine hydroxylase (AB151, Chemicon) and theneurotransmitters or neurotransmitter-related proteins vesicularacetylcholine transporter (AB 1578, Chemicon), neuropeptide Y (RIN7172,Peninsula Lab Inc.), substance P (RPN1572, Amersham Inc.), etc., asshown in FIG. 3, revealed cells approximately 2-4% positive for each,thereby also indicating the presence of neurotransmitter-producingneural cells.

Neural cells were induced by this procedure, and at this stage 2.9±0.5%of the total differentiation-induced neural cells exhibited reaction fortyrosine hydroxylase, a marker for dopaminergic neurons, as shown at theleft of the graph of FIG. 5. Also, as shown at the left of the graph inFIG. 7, 1.78±0.75% of the total differentiation-induced neural cellsexhibited reaction for vesicular acetylcholine transporter, a marker foracetylcholinergic neurons which are typically motor neurons.

Example 2 Induction of Dopaminergic Neurons

The differentiation-induced neural cells were then cultured in MinimumEssential Medium Alpha Eagle Modification (M4526, Sigma Co.) containing10% fetal bovine serum (14-501F, Lot #61-1012, BioWhittaker Co.), withfurther addition of 50 ng/ml of glial derived neurotrophic factor (GDNF)(human recombinant GDNF, #450-10, Peprotech EC Ltd.), 5 μM of forskolin(344273, Calbiochem), 10 ng/ml of basic fibroblast growth factor(100-18B, Peprotech EC, Ltd.) and 5 ng/ml of platelet-derived growthfactor-AA (396-HB, Peprotech EC Ltd.).

As a result of this procedure, the dopaminergic neurons exhibitingreaction for tyrosine hydroxylase increased dramatically to 17.2±5.1% ofthe total neural cells (see right of graph in FIG. 5). As shown in thephotograph of FIG. 4, the proportion of tyrosine hydroxylase proteinstained green with FIPC increased dramatically after GDNF treatment.

Example 3 Induction of Acetylcholinergic Neurons

The differentiation-induced neural cells of Example 1 were cultured inMinimum Essential Medium Alpha Eagle Modification (M4526, Sigma Co.)containing 10% fetal bovine serum (14-501F, Lot #61-1012, BioWhittakerCo.), with further addition of nerve growth factor (2.5 S NGF, #T002A,Takara), 5 μM of forskolin (344273, Calbiochem), 10 ng/ml of basicfibroblast growth factor (100-18B, Peprotech EC, Ltd.) and 5 ng/ml ofplatelet-derived growth factor-AA (396-HB, Peprotech EC Ltd.).

As a result of this procedure, the acetylcholinergic neurons exhibitingreaction for vesicular acetylcholine transporter increased dramaticallyto 20.5±0.05% of the total neural cells (see right of graph in FIG. 7).As shown in the photograph of FIG. 6, the proportion of vesicularacetylcholine transporter protein stained green with FIPC increaseddramatically after NGF (neurotrophin (NTs) treatment.

Example 4 Skeletal Muscle Induction

Stromal cells were extracted from the bone marrow of adult rats (Wistarrats) and cultured. The medium used was Minimum Essential Medium AlphaEagle Modification (M4526, Sigma Co.) containing 20% fetal bovine serum(14-501F, Lot #61-1012, BioWhittaker Co.).

After subculturing to four generations, 3 μmol/l of 5-azacytidine wasadded when the cells reached 80-90% confluence, and culturing wascontinued for 24 hours.

The medium was then switched with one containing 5 μM of forskolin(344273, Calbiochem), 10 ng/ml of basic fibroblast growth factor(100-18B, Peprotech EC, Ltd.) and 5 ng/ml of platelet-derived growthfactor-AA (396-HB, Peprotech EC Ltd.) and 200 ng/ml of heregulin(396-HB, R&D Systems), and culturing was continued for another 7 days.

The Notch intracellular domain gene was then introduced in the samemanner as Example 1.

On the day following introduction, G418 sulfate (83-5027, Gibco BRL) wasadded to a concentration of 200 ng/ml and introduced cells were selectedfor 10 days.

After restoration of the cell population to approximately 100%confluence, non-treated bone marrow stromal cells without the introducedgene were added to the medium and co-cultured therewith.

After three days, 5 μM of forskolin (344273, Calbiochem) was added.After several more days, the cells fused into locally appearingpolynucleated skeletal muscle cells (see FIG. 8), in an increasingmanner with time (FIG. 9). The skeletal muscle cells were observed witha confocal laser microscope, as seen in FIG. 10. Expression of myogeninand Myf5 mRNA in the cells was confirmed by RT-PCR. Electron microscopeobservation revealed myofibers characteristic of skeletal muscle cells.

Example 5 Therapeutic Effect of Dopaminergic Neurons Obtained byDifferentiation Inducing Method of the Invention when Transplanted intoStriata of Rat Parkinson's Disease Models

We examined the effect of transplanting dopaminergic neurons obtained bythe differentiation inducing method of the invention into ratParkinson's disease models. Injection of 6-OHDA (6-hydroxydopamine) intorat brain substantia nigra has already been established as a method ofcreating Parkinson's models, and these models were used for the presentexperiment (Svendsen et al., Exp. Neurol. 137:376-388(1996); Svensen etal., Exp. Neurol. 148:135-146(1997)). Administration of apomorphine tosuch rat models is known to provoke rotational movement, with increasingrotations suggesting deterioration and reduced rotations suggestingimprovement.

As shown in the top graph of FIG. 11, with grafting of induced neuralcells into striata, the number of rotations per minute during a 9-weekobservation period was approximately unchanged as compared toimmediately after grafting. In the absence of treatment, the number ofrotations per minute tended to gradually increase (not shown), andtherefore the level slope indicated that at least aggravation wasprevented.

As shown in the bottom graph of FIG. 11, with grafting of induceddopaminergic neurons into the striata, the number of rotations perminute began to decrease from the first week after grafting, and inapproximately half of the animals, a very notable improvement was foundwith the number of rotations per minute reaching zero or only 1 or 2after 9 weeks. (The two cases in the bottom graph of FIG. 11 whichexhibited more than 8 rotations/minute after 9 weeks were thought torepresent grafting failures and were excluded from the evaluation.)

In order to investigate the type of cells into which the dopaminergicneurons of the invention injected (grafted) into the striata haddifferentiated, the striatal tissue was extracted after 10 weeks andslices thereof subjected to an immunohistochemical examination.

The gene for green fluorescent protein (GFP) which emits greenfluorescent light was incorporated into the chromosomes of bone marrowstromal cells using a retrovirus. Thus, as seen in the immunofluorescentphotographs shown in FIG. 12, the neural cells induced to differentiatefrom bone marrow stromal cells, and therefore the dopaminergic neuronsgrafted into striata, emit green fluorescent light.

Also, red light emission was used for neurofilament as a marker forneural cells, tyrosine hydroxylase as a marker for dopaminergic neurons,GFAP as a marker for astrocytes (glial cells) and O4 as a marker foroligodendrocytes (glial cells).

Thus, superposition of green light by GFP and red light by theaforementioned markers produces yellow light, for distinction of thetype of cells that the grafted dopaminergic neurons had become 10 weeksafter grafting.

As seen in FIG. 12, almost all of the striata-grafted cells haddifferentiated to neural cells but not to glial cells 10 weeks aftergrafting. Also, judging from the considerable number of tyrosinehydroxylase-positive neural cells (i.e. dopaminergic neurons), it isconcluded that the in vitro differentiation inducing method of thepresent invention increased the proportion of dopaminergic neurons to17.2±5.1% of the total neural cells, and that the aforementionedgrafting further increased this proportion.

FIG. 13 is a composite of magnified immunofluorescent photographsshowing coloration of tyrosine hydroxylase.

This procedure demonstrated that in these rat Parkinson's diseasemodels, grafting of dopaminergic neurons obtained by the differentiationinducing method of the invention into striata dramatically improved thesymptoms of Parkinson's disease.

The following are the experimental protocols which were used in Examples6 to 11 below.

Experimental Protocols

Culturing of Bone Marrow Stromal Cells

Isolation of MSCs from Wistar rat bone marrow has been described inprevious publications by the present inventors⁽⁴⁾. Human MSCs wereobtained from a commercially available source (PT-2501, BioWhittaker,Walkersville, Md.) and a healthy donor (obtained in conformity with theguidelines of the Ethics Committee of Kyoto University Graduate Schoolof Medicine). The human MSCs were isolated by a previously describedmethod⁽³⁾. The cells were cultured in alpha-MEM (M-4526, Sigma, St.Louis, Mo.) containing 10% fetal bovine serum (FBS).

FACS Analysis

Rat MSCs were incubated with FITC-labeled mouse anti-CD34 (Santa CruzBiotechnology Inc., Santa Cruz, Calif.), anti-CD54, -CD90 and -CD11b/cor hamster anti-CD29 (PharMingen, San Diego, Calif.). Controls wereincubated either with FITC-labeled anti-mouse or anti-hamster IgG, ornon-immune mouse serum. For human MSCs there were usedphycoerythrin-labeled mouse anti-CD34, -CD29, -CD90, -CD54, -CD11c and-von Willebrand factor. Controls included cells stained withphycoerythrin-labeled anti-mouse IgG. The data were acquired andanalyzed on FACScalibur with CellQuest software (Becton Dickinson,Franklin Lakes, N.J.).

Plasmids

Numbering of Notch1 was according to Weinmaster et al. (1991) 39. cDNAfor the m-Notch 1 intracellular domain NICD (starting at amino acid 1703and terminating at the 3′ untranslated sequence), TM (amino acids1747-2531), M2 (modified from TM by mutation of two amino acids Ala-Ala(1992 and 1993) to Glu-Phe) (NICD, TM and M2 provided by Dr. MasashiKawaichi)^((17,34)), mNIC Δ3′ (amino acids 1846-2477, provided by Dr.Jeffery Nye)⁽³⁵⁾, RAMIC (amino acids 1703-1969, obtained from NICD cDNAby digestion with NotI and AccIII) and TADIC (amino acids 2192-2531,obtained from NICD cDNA by digestion with XhoI and XbaI) were subclonedinto pCI-neo mammalian expression vector (Promega, Madison, Wis.).Luciferase reporter plasmids of 3-PGDH (both full length and M1965) wereprovided by Dr. Shigeki Furuya⁽¹⁹⁾, NeuroD by Ming-Jer Tsai⁽⁴⁰⁾, andGFAP promoter by Caleb E Finch⁽⁴¹⁾. MSCs were transfected with theseplasmids using lipofectamine 2000 (Invitrogen, Carlsbad, Calif.) andselected by G418 according to manufacturer's instruction.

Neural Induction Experiment

For trophic factor induction, subconfluent cultures of NICD-transferredMSCs were incubated in alpha-MEM containing 10% FBS, 5 μM FSK(Calbiochem, La Jolla, Calif.), 10 ng/ml bFGF (Peprotech, London, UK)and 10 ng/ml CNTF (R&D Systems, Minneapolis, Minn.). For GDNF treatment,50 ng/ml of GDNF (Peprotech) was administered into alpha-MEM culturemedium containing 10% FBS.

Brd-U Labeling

After trophic factor induction (5 days), Brd-U (10 μM) was added to theculture medium and culturing was carried out for 24 hours. Cells werethen fixed with 4% paraformaldehyde in PBS and double labeled forMAP-tab and Brd-U, prior to TOTO-3 (Molecular Probes) counter staining.

RT-PCR Analysis

Total cellular RNA was isolated using an SV total RNA isolation system(Promega). To analyze relative expression of different mRNAs, the amountof cDNA was normalized based on the signal from ubiquitously expressedβ-actin mRNA. PCR was performed using standard protocols with Taqpolymerase (Sigma). Cycling parameters were denaturation at 94° C. for30 sec, annealing at 54-68° C. for 1 min depending on the primer, andelongation at 72° C., with 35 cycles.

Immunocytochemistry

The specific procedure has been previously described⁽⁴⁾. Antibodies toGLAST were provided by Dr. Masahiko Watanabe⁽¹⁸⁾, and 3-PGDH by Dr.Shigeki Furuya⁽¹⁹⁾. The following primary antibodies were purchasedcommercially: nestin (1:500, PharMingen), MAP-2ab (1:250, Sigma),neurofilament-M (1:200, Chemicon, Temecula, Calif.), β-tubulin isotype 3(1:400, Sigma), TuJ-1 (1:100, Babco, Richmond, Calif.), GFAP (1:1, DAKO,Carpinteria, Calif.), O4 (1:20, Boehringer Mannheim, Germany), GalC(1:30, Chemicon), GABA (1:1000, Sigma), serotonin transporter (1:200,Chemicon), vesicular acetylcholine transporter (1:100, Chemicon),glutamine (1:100, Chemicon), neuropeptide Y (1:1000, PeninsulaLaboratories Inc., Belmont, Calif.), TH (1:200, Chemicon), VIP (1:500,Incstar, Stillwater, Minn.), CGRP (1:1200, Amersham, Buckinghamshire,UK), SP (1:500, Amersham), DAT (1:200, Chemicon). Cells were incubatedwith Alexa Fluor 488- or 546-conjugated secondary antibodies, and TOTO-3iodide counter staining was performed. The cells were examined under aconfocal laser scanning microscope (Radians 2000, Bio-Rad,Hertfordshire, UK).

Reporter Assays

Cells were transfected using lipofectamine 2000 (Invitrogen) accordingto the manufacturer's instruction. Forty-eight hours after transfection,cells were assayed for Firefly and Renilla luciferase activities using adual luciferase assay kit (Promega). Firefly luciferase values werecorrected for transfection efficiency by including plasmids expressingRenilla luciferase.

Western-Blot Analysis

Cell lysates were prepared and 50 μg of lysate proteins wereelectrophoresed on 5% and 10% SDS-polyacrylamide gel. Antigens to MAP-2(1:500, Chemicon), GFAP (1:500, Dako) and TH (1:1000, Chemicon)antibodies were detected using alkaline phosphatase.

Electrophysiological Methods

Currents were measured at room temperature (20-25° C.) with a CEZ-2300(Nihon Kohden, Tokyo, Japan) patch-clamp amplifier. Data acquisition andstimulation were controlled with the pClamp 6.0 software (AxonInstruments, Inc., Foster City, Calif.). Signals were filtered at 5 kHzand sampled at 10-50 kHz. Experiments were performed in a whole-cellpatch-clamp configuration using pipettes (borosilicate glass, Narishige,Tokyo, Japan) with resistance values in the range of 4-8 MΩ. Forrecording of delayed rectifier potassium currents, the standardextracellular solution contained (mM) NaCl (150), KCl (4), CaCl₂ (2),MgCl₂ (2), glucose (10) and Hepes (10) (pH 7.4 with NaOH). The standardpipette solution was (mM) KCl (130), MgCl₂ (5), EGTA (10), and Hepes(10) (pH 7.4 with KOH).

Analysis of Parkinson Disease Model Rats

A procedure for creating this disease model has been described in aprevious report⁽⁴⁵⁾. In brief, adult male Wistar rats (weighing 250-300g) were anesthetized with sodium pentobarbital (40 mg/kg,intraperitoneal), and then 6-OHDA solution (8 μg/4 μl of 0.1%ascorbate-saline) was injected into the left medial forebrain bundle(A/P=−4.4 mm; L=+1.1 mm from bregma, V=−7.7 mm from dura).

Prolonged contralateral rotation was used as a target behavior, and ratsshowing an average of fewer than 6 rotations per minute for the first 30minutes after apomorphine administration (0.8 mg/Kg, subcutaneous) wereexcluded. 1×10⁵ cells/8 μl were grafted into the lesioned striatum atthe following coordinates: A/P=+0.5 mm; L=+3.0 mm from bregma, andV=−4.5 mm. The number of animals were 5 in the MSC group, 6 in the N-MSCgroup and 10 in the G-MSC group.

For immunohistochemistry of grafted striata (G-MSC group 10 weekspost-operation), glia sections were incubated with antibodies againstneurofilament-M, TH, DAT, GFAP and O4. These were then detected byAlexafluor 546-labeled secondary antibodies (Molecular Probes), prior toTOTO-3 iodide counter staining.

For human MSC transplantation, 5 animals were grafted andimmunosuppressed by subcutaneous injection of FK506 (1 mg/kg, Fujisawa,Osaka, Japan) once a day. Four weeks after transplantation, apomorphineinduced rotation was measured. For dopamine measurement in HPLC, 1 mmthick coronal brain slices were obtained (A/P+2.5 mm to −1.5 mm frombregma; 4 slices total), separated at the midline, and each side wascultured separately in alpha-MEM containing 10% FBS. After 24 hours, theculture media were collected and provided for HPLC analysis by SRLCommunication and Health, Tokyo, Japan. All animal experiments wereapproved by the Animal Care and Experimentation Committee of KyotoUniversity Graduate School of Medicine.

Statistical Analysis

Data were expressed as mean±SEM. Data were compared using ANOVA withpairwise comparisons by the Bonferroni method. P values of <0.05 wereregarded as significant, and <0.01 as highly significant.

Example 6 Identification of MSCs

The rat and human MSCs were used for the next experiment. The rat MSCs(Wistar) were isolated by a previously described method and cultured⁽⁴⁾.The human MSCs were obtained from a healthy donor or purchased from acommercial source (BioWhittaker).

The cell surface markers were evaluated on the rat MSCs and human MSCsusing fluorescent activated cell sorting (FACS). The MSCs expressed CD29(β-integrin), CD90 (Thy-1) and CD54 (ICAM-1), but not CD34 (hemopoieticstem cell marker), CD11b/c (macrophage-related marker) or von Willebrandfactor (human endothelial cell marker, data not shown) (FIG. 14 a). Thisresult matched previous reports^((3,11)). Similar results were obtainedby immunocytochemical examination (FIGS. 14 b-f). Lipogenic,chondrogenic and osteogenic differentiation from both the rat and humanMSCs were confirmed according to the method described in Pittenger etal. (1999)⁽³⁾. This indicated that the cells were a mesenchymal source(data not shown).

Example 7 Effect of NICD Transfection on MSCs

MCD was transfected into the MSCs, since Notch signaling activity isfound in the intracellular domain of the Notch protein and deletionsthat remove the extracellular domain can elicit a constitutively activeform of Notch⁽¹⁶⁾. MCD comprises a sequence coding for a smallextracellular domain portion, the transmembrane region and the entireintracellular domain of mouse Notch⁽¹⁷⁾, and was provided by Dr.Kawaichi of the Nara Institute of Science and Technology. The fragmentwas subcloned into pCI-neo, a mammalian expression vector containing theCMV promoter, and then transfected into the MSCs by lipofection andsubsequent selection of G418.

Since the Notch extracellular and intracellular domains were detected,the non-treated MSCs expressed small amounts of endogenous Notch.However, the MCD-transfected MSCs predominantly expressed only MCD andthe extracellular domain was not detected (FIG. 15 a).

The glutamate transporter GLAST and 3-phosphoglycerate dehydrogenase(3PGDH) are present in neural stem cells (NSC) and radialglia^((18,19)). These are thought to be lineally related to stem cells,and may serve as a source of neurons during embryogenesis⁽²⁰⁾.Bromodeoxyuridine (Brd-U)-positive NSCs in the dentate gyrus of adultmouse hippocampus were almost invariably immunopositive for 3PGDH⁽¹⁹⁾.After transfection of NICD, the rat MSCs upregulated transcription andexpression of both of these molecules as well as nestin, a known markerfor NSC and neural progenitor cells (NPC)⁽²¹⁾. The non-treated MSCsexhibited almost no expression of GLAST or 3PGDH, but a very smallfraction of cells were positive for nestin (0.74±0.1%). After MCDtransfection, however, these cells upregulated GLAST, 3PGDH and nestin(4.92±1.0%, p<0.01) (FIGS. 15 b-g). In a luciferase promoter assay,5′-flanking full length (nucleotides −3472 to −1) and 5′-flanking M1965(−1792 to −1) 3PGDH activities (both reported to be active in radialglia and neuroepithelial stem cells⁽¹⁹⁾) were significantly increased inthe rat MSCs after MCD transfection (p<0.01) (FIG. 15 h). (The promoterswere provided by Dr. S. Furuya, Brain Science Institute, RIKEN).

In vertebrates, NSC and neural crest stem cells adopt a glial fatethrough inhibition of neural differentiation^((13,14,16)). The presentinventors have confirmed that insertion of NICD into rat NSCs generatesGFAP-positive astrocytes, but very few GFAP-positive cells werediscovered in the MCD-transfected MSCs (data not shown). On the otherhand, it has been reported that introduction of activated Notch1 intomouse forebrain promotes radial glia identity during embryogenesis⁽¹⁵⁾.Since the MSCs expressed NSC and NPC related markers after introductionof NICD, it is plausible that MCD transfection caused the MSCs to changetheir phenotype to one resembling NSCs and/or NPCs.

Neural Induction in MCD-Transfected MSCs

The present inventors investigated the conditions necessary toselectively generate neural cells from NICD-transfected MSCs. Wetherefore tested various factors known to act on neurogenesis⁽²²⁾(neurotrophins, leukemia inhibitory factor, bFGF and CNTF) andforskolin. We found that the most efficient condition for specificinduction of neural cells was simultaneous introduction of FSK, bFGF andCNTF. (Hereinafter referred to as “trophic factor introduction”throughout the present specification.)

Following NICD transfection into rat MSCs, culturing of the cells to60-70% confluence and introduction of three trophic factors(FSK+pFGF+CNTF), 96.5±0.6% of the cells were MAP-2ab positive after 5days (FIG. 16, FIGS. 17 a-d). The present inventors observedMAP-2ab-positivity rates of 73.2±5.1% with bFGF alone and 87.5±3.1% and83.6±3.4% when FSK and CNTF were also added. This difference was notsignificant (p>0.05)(FIG. 16). FSK and CNTF respectively produced ratesof 29.2±5.4 and 4.3±1.9% alone (p<0.01) and 11.4±2.4% together (FIG.16).

The induction of MAP-2ab cells by trophic factors was most likely causedby inhibition of glial and other cell differentiation from MSCs ratherthan by specific killing of non-neural cells, because almost no deadcells were observed by TOTO-3 nuclear staining following trophic factorinduction (data not shown).

Trophic factor induction by itself, or after insertion of a pCI-neocontrol vector without NICD, resulted in no recognizable neuralphenotypes (FIG. 16). Therefore it would seem that NICD transfection iscritical for neural induction of MSCs.

Characterization of MSC Neural Cells

Neural cells derived from the aforementioned rat and human MSCs showeddistinct morphological features characteristic of neurons, includingneurite-like processes with abundant varicosities, and expressed typicalneural markers such as neurofilament-M, β3-tubulin and Tuj1 (FIGS. 17a-g). Nestin-positive cells, though few, could also be recognized(2.03±0.7%)(data not shown). Induced neural cells were unable toproliferate when subcultured after trypsin treatment. Brd-Uincorporation studied 5 days after trophic factor induction showedminimal labeling of MAP-2ab positive cells (FIG. 17 h), suggesting thatthese neural cells are mitotically terminated.

MAP-2ab was not detected by Western blotting in non-treated MSCs but wasfound after trophic factor induction (FIG. 17 l(1)).

A developmental rise in delayed rectifier potassium current isassociated with the maturation of cell excitability and neuraldifferentiation⁽²³⁾. The present inventors investigated this property inthe induced neural cells by using the voltage clamp method. An outwardlyrectified K+ current was elicited by positive voltage steps in inducedMSCs derived from both rats and humans. The amplitude of this currentwas dramatically higher than that in non-treated MSCs (FIGS. 17 m-q).The present inventors also investigated resting membrane potential undercurrent clamp conditions immediately after whole-cell configuration wasformed. Resting membrane potentials were lower among neural cells thanin non-treated MSCs (−50 to −60 mV and −30 to −40 mV respectively).These neurophysiological properties induced in MSCs resemble those ofmature neurons.

In checking for glial cells, the present inventors performedimmunocytochemistry using GFAP as a marker for astrocytes, andgalactocerebroside (GalC) and O4 as markers for oligodendrocytes. Nomarker-positive glial cells were detected after trophic factor inductionof rat or human MSCs (FIGS. 17 i-k). This was confirmed by Westernblotting (FIG. 17 l(2)). To further confirm specificity of neuralinduction, the present inventors measured the promoter activities ofNeuroD and GFAP. In non-treated rat MSCs, the rates for NeuroD and GFAPwere 67.2±15.3 and 5.16±1.36, respectively. Following trophic factorinduction, however, NeuroD activity increased significantly to132.7±20.9 while GFAP decreased to 0.63±0.22 (FIG. 18). These resultsindicate that only neural cells were specifically induced fromNICD-transfected MSCs after trophic factor induction.

Generation of TH-Positive Cells

Neural function is closely related to cell type-specificneurotransmitters. The present inventors therefore performedimmunocytochemical examination of neurotransmitters and related proteinsafter trophic factor induction (FIG. 19 a). GDNF is known to be involvedwith the generation and development of midbrain dopaminergicneurons⁽³⁶⁾. The present inventors also examined whether administrationof GDNF induces neural MSCs to increase their proportion of tyrosinehydroxylase (TH)-positive cells. This percentage increased from 3.9±0.6%after trophic factor induction alone up to 41.0±14.1% followingadministration of GDNF (FIGS. 19 a˜c). GDNF also induced expression ofNurr-1, which is a transcription factor that has a role in thedifferentiation of midbrain precursors into dopaminergic neurons⁽³⁷⁾(FIG. 19 d). Western blotting further confirmed these results (FIG. 19e).

Transplantation of Neural Cells to Parkinson's Disease Model Rats

In order to explore the ability of MSC derived neural cells to surviveand function in vivo, both rat and human cells were transplanted intothe striata of Parkinson's disease model rats. Unilateral administrationof 6-OHDA into the medial forebrain bundle selectively destroysdopaminergic neurons in the substantia nigra, thus providing a usefulmodel of Parkinson's disease. Three types of rat MSCs labeled with greenfluorescent protein (GFP)⁽⁴⁾ were transplanted: 1) non-treated (MSCgroup), 2) after trophic factor induction into neural cells (N-MSCgroup), and 3) GDNF administration after induction (G-MSC group).Animals received implantation of 1×10⁵ MSCs ipsilateral to the lesionedstriatum. Apomorphine-induced rotational behavior was examined for 10weeks following cell implantation. The MSC group showed a rotationalbias away from the lesioned side which persisted, whereas the N-MSCgroup showed slight recovery over time. In contrast, the G-MSC groupdemonstrated significant recovery from rotation behavior (FIG. 19 f).The transplanted animals were followed up to 16 weeks, with no tumorformation observed in the brain.

Ten weeks following grafting the brains were examined histologically,including immunohistochemistry. Grafted striata showed GFP-positivecells, while transplanted cells were positive for neurofilament and, ina few cases, showed labeling with anti-GFAP or anti-O4 antibodies. Manyof the transplanted cells were also positive for TH and dopaminetransporter (DAT)(FIGS. 19 g˜k). The percentage of GFAP-positive cellsamong GFP-labeled MSCs was 2.5±1.4%, while the percentages of TH- andDAT-positive cells were 45.7±4.2% and 30.7±0.9%, respectively. In serialsections of the G-MSC group, grafted cells were found to migrate andextend into the host striatum (FIG. 19 l). Approximately 3.4×10⁴ cells(34%) were counted in the striatum.

Human GDNF-treated neural MSCs were similarly transplanted into thestriata of 6-OHDA-lesioned rats. The animals were immunosuppressed withFK 506 daily, and rotation behavior was recorded at 4 weeks. Graftingresulted in significant improvement in rotational behavior (meanrotation index, post/pre-operation, was 0.44±0.2)(FIG. 19 m). Thecapacity of grafted human MSCs to synthesize and release dopamine wasassessed by measuring dopamine concentration in the culture medium ofslices of transplanted brain by high-performance liquid chromatography(HPLC). Brain slices were separated at the midline into grafted andintact sides and cultured separately. The dopamine concentration in theculture medium from each side was measured and the ratio of lesioned tointact side was calculated. Sham-operated rats showed a ratio of0.57±0.01 (n=3) in contrast to the grafted animals' ratio of 0.67±0.04(n=3). This was consistent with an increase in dopamine release (p=0.04)with transplantation. These results suggest that neural cells inducedfrom human MSCs were able to synthesize and release dopamine in lesionedrat striata.

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What is claimed:
 1. A cell culture comprising bone marrow stromal cells,wherein at least one bone marrow stromal cell in the culture comprisesexogenous nucleotide sequences, wherein the exogenous nucleotidesequences encode a notch intracellular domain but do not encodefull-length Notch protein.
 2. A cell culture comprising bone marrowstromal cells, wherein at least one bone marrow stromal cell in theculture expresses a notch intracellular domain encoded by an exogenouspolynucleotide.
 3. A cell culture comprising: (a) bone marrow stromalcells, wherein at least one bone marrow stromal cell in the culturecomprises exogenous nucleotide sequences, wherein the exogenousnucleotide sequences encode a notch intracellular domain but do notencode full-length Notch protein; and (b) a cyclic adenosinemonophosphate (cAMP)-increasing agent or a cAMP analogue and/or a celldifferentiation stimulating factor.
 4. A cell culture comprising:(a)bone marrow stromal cells, wherein at least one bone marrow stromalcell in the culture expresses a notch intracellular domain encoded by anexogenous polynucleotide; and (b) a cyclic adenosine monophosphate(cAMP)-increasing agent or a cAMP analogue and/or a cell differentiationstimulating factor.